Method for preparing human immunoglobulin concentrates for therapeutic use

ABSTRACT

The invention concerns a method for preparing human immunoglobulin concentrates for therapeutic use, from plasma or a plasma fraction. The method comprises pre-purification and a single anion-exchange chromatography carried out at alkaline pH, thereby enabling the immunoglobulins to be retained on the chromatographic support and fractionated. The method enables to obtain IgG, IgA and IgM concentrates.

[0001] The invention relates to a process for preparing humanimmunoglobulin concentrates for therapeutic use, from plasma or afraction of human plasma. The process enables immunoglobulins G (IgG),immunoglobulins A (IgA) and immunoglobulins M (IgM) to be obtained.

[0002] The use of human plasma fractions enriched with immunoglobulinsfor the treatment of various infections or congenital deficiencies hasbeen known. since the development of the ethanol precipitation processby Cohn (Cohn et al. 1946, J. Am. Chem. Soc. 68, 459 ; Oncley et al.,1949, J. Am. Chem. Soc. 71, 541). As the therapeutic indications forimmunoglobulins have grown in number, there is a need for a productoffering ever increasing performance and purity.

[0003] The complexity of the structure of immunoglobulins (fourpolypeptidic chains joined by disulphide bonds) and the variety ofantibodies present in the mixture of the plasma of several thousanddonors are not presently factors favouring the biotechnologicaldevelopment of immunoglobulins. Although some monoclonal antibodies areproduced by genetic engineering, their extreme specificity represents adrawback for therapeutic applications in which polyreactivity appearsnecessary.

[0004] In addition, numerous pathologies, in particular of autoimmuneorigin, are presently treated using IgG concentrates. This widespreaduse has led to a shortage in Europe and the United States over the lastfew years.

[0005] Furthermore, in these same pathologies, the efficiency ofIgM-enriched preparations has recently been demonstrated (Hurez et al.Blood 90, 1997, 4004-4013), but there exists no preparation fortherapeutic use that is sufficiently purified and that has a sufficientIgM concentration.

[0006] This is why the Applicant has devoted itself to the developmentof a new process for the preparation of human immunoglobulins. Theprocess can be applied to a pool of serums (from at least 10 000donors), which ensures the presence of all the antibodies normallypresent throughout the population of a chosen region, or to hyperimmuneserums selected for their specific immunoglobulin content. The processfurther allows the preparation of IgA and IgM concentrates.

[0007] Numerous variants of the original Cohn process have beendescribed. They propose, in addition to the selective precipitation ofthe proteins with ethanol, various additional treatments such asprecipitation with polyethylene glycol, the gentle treatment withproteolytic enzymes, etc., intended to eliminate the aggregates ofimmunoglobulin polymers (liable to activate the complement system and tolead to anaphylactic reactions).

[0008] An alternative approach to ethanol precipitation is described bySteinbuch et al. (Rev. Franc. Et. Clin. et Biol. 1969, XIV, 1054) ; thisapproach uses precipitation with octanoic acid. This acid precipitatesmost of the proteins in the plasma and leaves the immunoglobulins in thesupernatant. Purification of these immunoglobulins is continued byadsorption (in “batches”) using an anion exchanger, DEAE-cellulose,which also leaves the immunoglobulins in the supernatant. The lafter isthen concentrated.

[0009] Various processes have also been developed for increasing thepurity of the products through the use of chromatographic separationprocesses. Those yielding the best performance (in particular EP 0 703922, WO 99/64462) include at least two successive chromatography steps,one using anion exchange and the other using cation exchange. Thespecificity of these processes is provided by the property of the anionexchangers of not adsorbing the immunoglobulins G, under conventionalconditions of implementation, but of fixing most of the other proteinsco-purified during the pre-purification steps.

[0010] Various purified IgG preparations are thus already available buttheir preparation processes still pose problems in terms of productivityand complexity of implementation on an industrial scale. These problemsare further increased by the need to include in the process viralinactivation, involving an additional step to eliminate the virucidalagents used.

[0011] In addition, all of the processes presently described have beendeveloped and optimised for the production of IgGs only.

[0012] The Applicant has found it to be possible to produce severalconcentrates of immunoglobulins by combining a pre-purification step anda single ion exchange chromatography step and thus implement a verysimple process that is highly productive and compatible with a virucidalsolvent-detergent treatment.

[0013] The process according to the present invention is thus applicableto blood plasma or to a fraction of blood plasma already enriched withimmunoglobulins, and it includes pre-purification through theprecipitation of lipidic and proteic contaminants and a single anionexchange chromatography, carried out at an alkaline pH, which allows theadsorption of the immunoglobulins on the anion exchange chromatographicmaterial.

[0014] Pre-purification is carried out by means of known precipitatingagents such as octanoic acid, tricalcium phosphate or bentonite.

[0015] After this pre-purification step and prior to chromatography, theprocess includes a step of viral inactivation, preferably usingsolvent-detergent, according to a known method.

[0016] At the end of this treatment, the pre-purified filtrate andsolvent-detergent mixture is subjected to chromatographic separationwhich is carried out using a DEAE, TMAE or QAE groups-grafted gel ofcross-linked polysaccharide or vinyl polymer. This chromatography stepincludes:

[0017] adjusting to a pH of 8.9 to 9.1 the solution that has undergonethe solvent-detergent treatment;

[0018] injecting it onto the chromatographic column previouslyequilibrated with buffer at a pH of 8.9 to 9.1, which permits adsorptionof the immunoglobulins and allows the non-adsorbed proteins to flowthrough into the effluent;

[0019] washing with the same buffer until all the non-adsorbed proteinsand the solvent-detergent mixture have been eliminated; and

[0020] eluting the immunoglobulins with a suitable buffer.

[0021] Said elution can be carried out either with a phosphate buffer ata pH between 4 and 7, and preferably, at a pH of 6.2, to elute theimmunoglobulins G, or sequentially:

[0022] initially, as above to elute the immunoglobulins G;

[0023] subsequently, using the same phosphate buffer to which has beenadded 100 to 175 mM NaCl, and preferably, 150 mM, at a pH of 6.1 to 6.3,to elute a fraction containing the IgAs and IgG4s.

[0024] The process can be continued by further elution with the samebuffer adjusted to a pH of 6 to 7 to which has been added 250 to 350 mMNaCl, and preferably, 300 mM, to elute the IgMs.

[0025] The immunoglobulins thus eluted are harvested, concentrated byultrafiltration and subjected to conventional sterile filtration, andthen to filtration through nanometric filters of a porosity decreasingfrom 100 to 50 and at 20 nanometers. This nanofiltration enables virusesresistant to the solvent-detergent treatment to be eliminated.

[0026] A pharmaceutically acceptable stabilising agent is added to theconcentrated and filtered solution of immunoglobulins, and the latter isthen packaged as a sterile solution, and optionally deep-frozen andfreeze-dried.

[0027] The following examples illustrate the invention without, however,limiting its scope.

EXAMPLE I

[0028] As a starting material, use is made of 1 kg of “I+II+III”precipitate obtained from plasma treated with ethanol according to theCohn method (already mentioned) or the Kistler and Nitschmann method(1962, Vox Sang. 7, 414).

[0029] This precipitate is resuspended in acetate buffer (sodiumacetate-acetic acid) at a pH of 4.7 to 4.9, with stirring, at 20° C.

[0030] Octanoic acid is added thereto up to a final concentration of 20g/l. The acid should be added slowly, at ambient temperature.

[0031] To the mixture is added a filtration aid, and the precipitate isseparated by filtration by means of a filter press.

[0032] The filtrate is recovered, clarified and concentrated byultrafiltration, then it is subjected to sterile filtration at 0.45 μmand 0.2 μm.

[0033] It is then subjected to a solvent-detergent viral inactivationtreatment as described by Neurath and Horowitz (U.S. Pat. No.4,764,369).

[0034] A Triton® X 100 (octoxinol 10)/TnBP mixture is used.

[0035] The mixture is adjusted to 64 g/l of proteins, at a pH of 6.5.Contact is maintained for 4 to 6 hours, at between 4 and 25° C. Then,the pH is adjusted to 9 (with NaOH).

[0036] The mixture is then subjected to an anion exchange chromatographyusing a column.

[0037] A chromatographic column is filled with TMAE-Fractogel® used asan anion exchange material, equilibrated with a glycine-NaCl mixture(glycine: 0.676 g/l—NaCl: 0.526 g/l) at a pH of 9.

[0038] The mixture is injected onto the column in a proportion of 50 gof proteins per litre of gel.

[0039] Once this has been done, the column is washed with glycine-NaClbuffer at a pH of 9 (the same as for equilibrating purposes). Thewashing is monitored by the optical density of the effluent at 280 nm.

[0040] After reaching the base line optical density, the column iseluted with phosphate buffer at a pH of 6.2 (disodium hydrogenphosphate—sodium dihydrogen phosphate).

[0041] The eluate, containing the immunoglobulins G, is adjusted to a pHof 4.5 and subjected to ultrafiltration using cassettes.

[0042] The solution is then subjected to sterile filtration at 0.22 μm,and then to nanometric filtration using three filters arranged in seriesand having decreasing thresholds of retention: 100, 50 and 20nanometers. Filtration is followed by ultrafiltration using a cassetteto concentrate the final solution to 120-150 g/l.

[0043] The analysis results for three pilot batches are given in thefollowing table. TABLE I PILOT BATCHES 00 ILP 043 PV 00 ILP 044 PV 00ILP 045 PV Polymers (%) 0.19 0.19 0.07 Dimers (%) 3.29 2.27 2.15Monomers (%) 96.52 97.54 97.78 IgG (g/l) 52.5 50.4 50.8 IgG1 (g/l) 31.629.9 29.2 IgG2 (g/l) 17.1 15.3 13.1 IgG3 (g/l) 1.19 1.07 0.96 IgG4 (g/l)0.52 0.61 0.56 IgM (μg/l) 310 459 1795 Anti-Hbs (IU/ml) 10.2 10.3 9.4

EXAMPLE II

[0044] As a starting material, use is made of 1670 g of “Precipitate II”obtained from 80 litres of plasma treated with ethanol according to theCohn method.

[0045] This variant of the process makes it possible, in particular, toturn to good account type II precipitates deep frozen and stored beforebeing subjected to further treatment.

[0046] This precipitate is resuspended in 9 kg of a water-NaCl mixture.The pH is adjusted to 6.2 with acetic acid.

[0047] 15 g of tricalcium phosphate are added thereto as an adsorbingagent (for contaminants of the lipidic type, kallikrein, etc.).

[0048] After one hour of contact, a filtration aid is added to themixture and the precipitate is separated by filtration by means of afilter press.

[0049] The filtrate is recovered, glycine (15 g/l) is added thereto, andis adjusted to a pH of 4.8 with acetic acid.

[0050] 80 g of bentonite are added thereto as an adsorbing agent (inparticular for the traces of activated clofting factors), with stirringfor 30 minutes.

[0051] A filtration aid is added to the mixture and the precipitate isseparated by filtration by means of a filter press.

[0052] The filtrate is recovered and concentrated by ultrafiltration.

[0053] The process is continued (by solvent-detergent treatment andchromatography), as in example I.

[0054] The analysis results for the three pilot batches are set out inthe following table. TABLE II PILOT BATCHES 286 287 321 Polymers (%) 0.50.5 <0.1 Dimers (%) 2.8 2.8 1.4 Monomers (%) 96.7 96.7 98.5 IgG (g/l)51.8 53.2 61.3 IgG1 (%) 62.5 62.6 64.4 IgG2 (%) 33.0 33.0 32.2 IgG3 (%)3.2 3.3 2.2 IgG4 (%) 1.2 1.2 0.8 IgA (mg/l) 26.4 21.5 12.6 IgM (μg/l)90.8 69.4 123 Anti-Hbs (IU/ml) 147 122 8.6

EXAMPLE III

[0055] The process is implemented in the same way as in example 1, butthe starting material is plasma (non precipitated with ethanol).

[0056] This variant is not advantageous from the productivity viewpointbut it is of interest (thanks to its small number of steps) in thetreatment of plasmas that present particularly a high rare antibodiescontent.

EXAMPLE IV

[0057] The process is implemented in the same way as in Example 1, butthe chromatographic column is sequentially eluted:

[0058] after the injection and the washing of the column, it is elutedwith a first phosphate buffer at a pH of 6.2 to elute the IgGs as inexample I;

[0059] next, it is eluted with the same buffer, to which has been added150 mM NaCl, which elutes an IgA and IgG4-enriched fraction.

[0060] IgG4s appear to play a part in protection from parasiticinfections and from allergies to pollens and mites.

[0061] Surprisingly, in their biochemical properties and their behaviourduring ion exchange, they resemble immunoglobulins A.

EXAMPLE V

[0062] The process is implemented in the same way as in example IV, but,after the second elution that produces the IgA-enriched fraction, 300 mMNaCl is added to the same buffer, which desorbs the IgMs. There is thusobtained a concentrated (80%) fraction of IgMs. These are concentratedand pharmaceutically acceptable stabilising agents are added thereto.

[0063] The production results for a batch of concentrated IgMs are setout in the following table. TABLE III (IgM batch 2000-97) Ig VolumeTotals IgM IgG IgA IgM Fraction (ml) (mg) (mg) (%) (%) (%) Pre-purifiedIg 2190 39957 2037 92.6 2.2 5.1 Fraction not adsorbed 3800 566.8 <32.386.5 <7.8 <5.7 on the column Eluate 1 (IgG) 2700 37374 <22.9 99.8 <0.1<0.1 Eluate 2 (IgA) 1350 3171 62.5 74.9 23.1 2 (0.15M NaCl) Eluate 3(IgM) 114.5 1775.1 1545.7 6.2 6.8 87.1 (0.3M NaCl)

1. Process for preparing human immunoglobulin concentrates fortherapeutic use, from blood plasma or a fraction of plasma enriched withimmunoglobulins, characterised in that it includes pre-purification byprecipitating lipidic and proteic contaminants and a single anionexchange chromatography, carried out at an alkaline pH, which allowsadsorption of the immunoglobulins on the anion exchange chromatographicmaterial.
 2. Process according to claim 1, characterised in that thepre-purification is carried out by means of known precipitating agentssuch as octanoic acid, tricalcium phosphate or bentonite.
 3. Processaccording to claim 1 or 2, characterised in that it comprises, afterpre-purification and prior to chromatography, a viral inactivationtreatment, preferably using solvent-detergent.
 4. Process according toany one of claims 1 to 3, characterised in that chromatography iscarried out using a DEAE, TMAE or QAE groups-grafted gel of cross-linkedpolysaccharide or vinyl polymer.
 5. Process according to any one ofclaims 1 to 4, characterised in that chromatography includes: the use ofthe solution that has undergone the solvent-detergent treatment; itsadjustment to a pH of 8.9 to 9.1; injecting it onto a chromatographiccolumn previously equilibrated with buffer at a pH of 8.9 to 9.1, whichpermits adsorption of the immunoglobulins and allows the non-adsorbedproteins to flow through into the effluent; washing with the same bufferuntil all the non-adsorbed proteins and the solvent-detergent mixturehave been eliminated; and eluting the immunoglobulins with a suitablebuffer.
 6. Process according to claim 5, characterised in that elutionis carried out in one step, with a phosphate buffer at a pH between 4and 7, and preferably, at a pH of 6.2, to elute the immunoglobulins G.7. Process according to claim 5, characterised in that elution iscarried out sequentially, initially, using a phosphate buffer at a pHbetween 4 and 7, and preferably, at a pH of 6.2, to elute theimmunoglobulins G subsequently, using the same phosphate buffer to whichhas been added 100 to 175 mM NaCl, and preferably, 150 mM, at a pH of6.0 to 6.3, to elute a fraction containing the IgAs and IgG4s. 8.Process according to claim 6 or 7, characterised in that it furtherincludes elution with the same buffer adjusted to a pH of 6 to 7 towhich has been added 250 to 350 mM NaCl, and preferably, 300 mM, toelute the IgMs.
 9. Process according to any one of claims 1 to 8,characterised in that, after elution of the immunoglobulins, these areharvested, concentrated by ultrafiltration and subjected to conventionalsterile filtration, and then to filtration through nanometric filters ofa porosity decreasing from 100 to 15 nanometers.
 10. Process accordingto any one of claims 1 to 9, characterised in that a pharmaceuticallyacceptable stabilising agent is added to the concentrated and filteredsolution of immunoglobulins, and the latter is then packaged as asterile solution, and optionally deep-frozen and freeze-dried.